Toxoplasma gondii infection in sheep from Romania

Background Toxoplasmosis is a widespread zoonosis caused by the intracellular protozoan parasite Toxoplasma gondii. Limited epidemiological information is available about the prevalence of T. gondii in sheep in Romania, and a high incidence would have implications for both the economy and public health. To our knowledge, no studies are available about the T. gondii strains circulating in lambs. The objective of this study was to assess the prevalence of T. gondii in sheep (serology), lambs (serology, bioassay, PCR) and sheep abortions (PCR) in Romania. Moreover, the study aimed to perform the genetic characterization of T. gondii isolates from lambs. Methods Serum samples collected from 2650 sheep (2067 adults and 583 lambs) were tested for anti-T. gondii antibodies (IgG) using a commercial ELISA kit. Likewise, 328 pairs of diaphragmatic muscle-serum samples were collected from lambs aged between 2 and 4 months. Lamb serum samples were analyzed using MAT for anti-T. gondii antibody detection. The diaphragm tissue samples from MAT-positive lambs (at a dilution ≥ 1:25) were bioassayed in mice. The T. gondii strains were genotyped using 15 microsatellites markers. Additionally, brain and heart samples from 76 sheep abortions were analyzed for T. gondii DNA by polymerase chain reaction (PCR) targeting the 529-bp repeat region (REP529). Results The results showed that more than half of the tested sheep were T. gondii seropositive (53.5%). The seroprevalence was significantly higher in adults (61.1%) than in lambs (26.4%). The seroprevalence of T. gondii infection in slaughtered lambs, by MAT, was 37.5% (123/328). There were bioassayed in mice 56 diaphragmatic tissues from 123 seropositive lambs. Toxoplasma gondii strains were isolated from 18 (32.1%) lambs intended for human consumption. All T. gondii strains were confirmed by PCR. Six strains were genotyped using 15 microsatellite markers and belonged to genotype II. Toxoplasma gondii DNA was detected in 11.8% (9/76) of sheep abortions. Conclusions The present study showed the presence of T. gondii in sheep in all the regions considered in the study. The high prevalence of T. gondii infection in sheep and lambs, demonstrated by serology, molecular analysis and bioassay, highlighted that there is an important risk of human infection in consuming raw or undercooked sheep/lamb meat. Graphical Abstract

Paștiu et al. Parasites & Vectors (2023) 16:24 Background Toxoplasma gondii, Phylum Apicomplexa, is a zoonotic parasite with worldwide distribution. The parasite has a heteroxenous life cycle with Felidae as definitive host (asexual and sexual stages in the intestinal epithelium) and warm-blooded animals, including humans, as intermediate hosts (asexual extraintestinal stage, mainly in the brain and muscles) [1]. The infective stages are represented by the sporozoites from sporulated oocysts, the bradyzoites from tissue cysts and the tachyzoites [2]. Human infections can be acquired by the ingestion of sporulated oocysts from water or vegetables and tissue cysts from raw or undercooked meat. Also, the congenital transmission of tachyzoites represents an important route of infection for the fetus.
Toxoplasmosis in sheep was first described by Hartley et al. [3] and Hartley and Marshall [4]. Although cats and wild felids are considered the predominant source of infection in sheep due to continuous contamination of pastures with T. gondii oocysts, congenital transmission should not be forgotten [5]. In general, most sheep become T. gondii seropositive after birth, < 2% are infected congenitally and < 4% of the positive ewes transmit the parasite transplacentally [6]. After infection with T. gondii, sheep develop humoral and cellularmediated immune responses against the parasite, which provides effective protection against the parasite during subsequent pregnancies [6]. However, T. gondii is a major cause of abortion and stillbirth in sheep worldwide, causing significant economic losses to breeders due to infertility, reproduction disorders, loss of offspring and reduction of milk production [7,8].
Furthermore, the infection of sheep with T. gondii can have important implications for public health. In countries with a temperate climate, between 30 and 63% of T. gondii infections in humans have been attributed to the consumption of raw or undercooked meat and/or meat products [9]. Sheep meat is recognized worldwide as a major source of toxoplasmosis [10]. A positive correlation has been demonstrated between the consumption of sheep meat and the prevalence of toxoplasmosis in humans [11]. The ingestion of raw or insufficiently heatprocessed meat contaminated with T. gondii may facilitate zoonotic transmission of the parasite, which can be present in almost all edible sheep tissues [12]. A recent study showed an uneven distribution of T. gondii cysts in muscle samples from lambs and indicated that even small amounts of meat (5-10 g) can have the potential to transmit T. gondii [13].
Besides congenital transmission, T. gondii can cause severe clinical symptoms in individuals with a deficient immune system, such as HIV/AIDS patients, oncology patients or transplant recipients. For these immunocompromised individuals, recent studies have demonstrated an increased risk of acquiring T. gondii infection [14].
In Romania, sheep breeding was one of the oldest occupations, and the phenomenon that defines the history of shepherding is transhumance. A country with a great tradition of and experience in sheep breeding, in 2020 Romania ranked third in the European Union, after Spain and Greece, in terms of small ruminants herds, with 11,189 million head [15]. The sheep are raised mainly for milk and meat production. Lamb meat is consumed especially during the Easter holidays.
Seroprevalence of anti-T. gondii antibodies in humans in northwestern and central Romania was 59.5%, higher in rural (63.6%) than urban areas (55.1%) [16]. The annual risk of infection is estimated to be 3-4.5% for children/ adolescences, 1-2% for adults and 5.5% for people aged up to 65 years old [16].
Considering the epidemiological situation of T. gondii in humans in our country, the economic importance of sheep toxoplasmosis, as well as the limited amount of data regarding the toxoplasmosis in sheep in our country, the purpose of this study was to estimate the prevalence of T. gondii infection in sheep and lambs in Romania. The study was divided in four substudies: (1) seroepidemiological study; (2) bioassay study; (3) study on T. gondii prevalence in aborted fetuses; (4) the genetic diversity of the isolated strains from lambs.

Seroepidemiological study
A total of 2650 blood samples were collected by puncture of the jugular vein or during slaughtering of randomly selected adult sheep (n = 2067; age = 1-6 years old) and lambs (n = 583; age = 2-8 months old). The sheep originated in 16 counties from four regions of Romania: central (Alba, Mureș, Brașov, Harghita, Covasna), northwestern (Bihor, Cluj, Sălaj, Satu-Mare, Maramureș, Bistrița-Năsăud), western (Arad, Timiș, Caraș-Severin, Hunedoara) and southeast (Buzău) (Fig. 1). The sheep were reared in herds with 50-1000 animals per unit. The sheep herds were allowed to graze from spring to autumn, and spring waters represented the water main source. The samples were collected with the consent of sheep breeders or of the abattoir veterinarian. The sheep breeders voluntarily participated in the study. Data regarding the age, county and region were collected (Table 1, Fig. 1). The samples were refrigerated and transported on the same day to the laboratory. The blood samples were centrifuged at 3000 rpm for 10 min, and the sera were stored at − 20 °C until further analysis.
Toxoplasma gondii-specific IgG antibodies were detected by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Chekit Toxotest Antibody ELISA, Idexx-Bommeli, Switzerland) following the manufacturers' instructions. The test results were interpreted as positive, doubtful or negative.

Bioassay study
During slaughtering, paired samples of diaphragmatic muscle (5-25 g) and blood were collected randomly from 328 lambs from seven counties from four regions of Romania: central (Mureș, Harghita, Covasna), northwestern (Cluj, Maramureș), western (Arad) and southeast (Buzău) (Fig. 1). The lambs were 2-4 months old and weighed 8-25 kg. They were slaughtered during the Easter holiday and intended for familial consumption. A questionnaire was prepared for collection of data regarding the age and region of origin of the animals ( Table 3). The samples were transported under refrigerated conditions to the laboratory. Serum samples were tested by modified agglutination test (MAT) within 24-48 h. Diaphragmatic tissue samples were kept under refrigerated conditions (4 °C) until bioassay in mice was performed.
The MAT for the detection of T. gondii-specific IgG antibodies was performed as previously described [17], using an antigen prepared from formalin-fixed whole RH strain tachyzoites (Reims, France). Each serum sample was serially twofold diluted. The threshold dilution was 1:6, and dilutions continued until the end of seropositivity. All serum samples with a titer ≥ 1:6 were considered positive.
The diaphragm tissue samples from lambs identified as seropositive by MAT at a dilution ≥ 1:25 were bioassayed on pathogen-free Swiss mice. The protocol described in the study of Paştiu et al. [18] was used. In brief, each diaphragm sample was minced and mixed (Knife Mill Grindomix GM 200, Retsch, Haan, Germany) with digestion solution (0.25% trypsin (93,613, Sigma-Aldrich, Saint Louis, MO, USA)/0.025% EDTA). The homogenate was incubated at 37 °C for 1.5 h and then filtered through gauze. The suspension was centrifuged at 1800×g for 10 min, the supernatant was discarded, and the pellet (digest) was washed with PBS, pH 7.2 (phosphate-buffered saline), by centrifugation three times. The digest   16:24 was mixed with 3 ml PBS containing 100 μl antibiotic solution (20,000 units penicillin/10,000 units streptomycin, P0781, Sigma-Aldrich) [19]. For each sample, 0.5 ml of digest was inoculated intraperitoneally in two Swiss mice. Twice daily, the mice were monitored. After 4 weeks, all the surviving mice were killed and bled, and the sera were tested by MAT for anti-T. gondii IgG-specific antibody detection. After death of the mice, their brains were recovered. If signs of acute toxoplasmosis (ruffled fur, diminished response to handling, state of prostration, severe weight loss and neurological signs) were observed for 3 consecutive days; the mice had been killed earlier. Mouse brains were mechanically homogenized and examined microscopically for brain cyst search and counting. For each brain, five glass slides were prepared. Also, the mouse brains were analyzed by conventional PCR (cPCR) targeting 529-bp repeat element (RE) [20]. The isolated strains of T. gondii were subsequently genotyped.

Toxoplasma gondii prevalence in aborted fetuses
A total of 76 aborted sheep fetuses (n = 67) and dead born lambs (n = 9) were collected and analyzed for the presence of T. gondii. Aborted fetuses, originated from two counties in central Romania (Alba, Sibiu) and two counties in northwestern Romania (Cluj, Bistriţa-Năsăud) ( Fig. 1), were brought by the sheep breeders for investigations at the Faculty of Veterinary Medicine, Cluj-Napoca. The aborted fetuses were expelled at different periods of gestation. Samples of heart (n = 76) and brain (n = 73) were collected from each aborted fetus. The samples were kept at -20 °C until processing by cPCR [20].

Polymerase chain reaction (PCR)
DNA was extracted from mouse brains and aborted fetus tissues (heart and brain) using a commercial kit (Isolate Genomic II DNA Kit, Meridian Bioscience, USA) according to the manufacturer's protocol. Positive and negative controls were represented by DNA obtained from T. gondii RH strain and tissue from free-pathogen Swiss mice, respectively. Toxoplasma gondii DNA was amplified by conventional PCR using the pair primers Tox 4 (5′ CGC TGC AGG GAG GAA GAC GAA AGT TG 3′) and Tox 5 (5′ CGC TGC AGA CAC AGT GCA TCT GGA TT 3′) that amplify the 529-bp RE (REP529, GenBank accession no. AF146527) as previously described [20]. Positive and negative controls and distilled ultrapure water were included in each run.

Statistical analysis
Point estimates and 95% confidence intervals (95% CI) for the anti-T. gondii antibodies and T. gondii DNA were calculated. These parameters were determined overall and by age group (lambs < 1 year; adults ≥ 1 year) and for each region (central, northwest, west, southeast). Also, the estimated age of lambs was recorded (between 2 and 8 months), and six groups were formed (2 months; 3 months; 4 months; 5 months; 6 months; 8 months). The difference in prevalence among groups was statistically analyzed using a Chi-square test of independence. A P-value of < 0.05 was considered statistically significant. Data were processed using EpiInfo 2000 software (CDC, Atlanta, GA, USA) (http:// wwwn. cdc. gov/ epiin fo).
Eleven of 18 strains were subjected to genotyping. Only two T. gondii strains were genotyped on all 15 microsatellite markers (TgRO-1LCj; TgRO-2LCj). The other four strains (TgRO-3LCj; TgRO-4LCj; TgRO-5LCj; TgRO-6LCj) were incomplete with between 11 and 14 obtained in microsatellite markers. The amount of DNA for the other five strains was inadequate for microsatellite genotyping. Toxoplasma gondii isolates shared the same type II genotype with the exception of the missing markers for the four incomplete strains ( Table 4, Fig. 3).

Toxoplasma gondii prevalence in aborted fetuses
Toxoplasma gondii DNA was detected in nine (11.84%; 95% CI: 5.56-21.29) of 76 tested aborted/dead fetuses. Both brain and heart samples collected from all the nine aborted fetuses were positive at PCR targeting the 529-bp repeat region. The positive samples belonged to aborted fetuses expelled in the first periods of gestation, and none of the fetuses born dead had presented T. gondii DNA.

Discussion
Toxoplasma gondii is responsible for toxoplasmosis, an important zoonotic parasitic diseases, with the potential for severe consequences in not only humans but also all intermediate hosts such as sheep.
The seroprevalence of toxoplasmosis in sheep varies between studies, countries and geographic areas. The seroprevalence of anti-T. gondii antibody IgG types in sheep from different European countries is variable: 89% in France [22], 87.4% in Belgium [23], 74% in Great Britain [24], 46.5% in Spain [25], 28% in Italy [26], 27.8% in The Netherlands [27] and 17.2% in Latvia [28]. Other factors that may lead to variations in the seroprevalence of T. gondii infection may be the serological tests used, the number of animals tested, the age of the animals and management system [12,29]. The presence of cats, a history of abortions in sheep in the last 12 months, breed, farm size, semi-intensive growing system, mineral salt supplementation and water origin are considered the risk factors for T. gondii infection in sheep [30,31].
The results of the present study showed that T. gondii infection in sheep is widespread in Romania (53.5%). Of the 2650 sera tested, 4.6% had a doubtful result on the ELISA immunoassay technique. Generally, after infection of sheep with T. gondii oocysts, IgG antibodies reach the maximum level after 2 months and persist at high titers for several months [1,32], which may explain some of these doubtful cases. The doubtful reaction can also be due to the common antigens at the level of surface proteins present in some parasites from the phylum Apicomplexa such as Neospora spp., Toxoplasma spp. and Hammondia spp., etc. [33].
Seropositivity in sheep increased in our study with age, adult sheep being exposed to T. gondii oocysts for a longer period than lambs [34]. In Romania, previous studies reported a seroprevalence of T. gondii antibodies in lambs and adult sheep by ELISA, ranging between 6.5 and 30.6% [35,36] and 36.3-62.99%, respectively [36,37]. Our study confirms that age is a risk factor for T. gondii infection, the seroprevalence obtained in adults (61.1%) being more than twice that in lambs (26.4%). We investigated lambs aged between 2 and 8 months. No 2-month-old lamb was T. gondii seropositive. The highest seroprevalence of anti-T. gondii antibodies were obtained in the 4-month group (46.2%). Then, the seroprevalence decreased in the 5-and 6-month-old groups and increased in the 8-month-old group. In ruminants, unlike primates, maternal IgG antibodies are transmitted through colostrum and not transplacentally, and they pass in the bloodstream 24-48 h after birth [38]. Moreover, maternal antibodies decline by 3 months of age, and they disappear by 5 months of age [34]. High antibody titer and age of lambs > 5 months old suggest an immune response caused by an active T. gondii infection and not transcolostrally acquired antibodies [34].
Furthermore, a significant statistical difference was noticed in the seroprevalence obtained between the different regions of our study. The differences between  Table 4 Genotypes of T. gondii isolates from sheep, pigs, goats and humans used in our study *The reference strains are available at the Toxoplasma Biological Resource Center (http:// www. toxoc rb. com) The TgRO-1LCj strain has been used previously as a reference strain under the name FR-OVI-ARI061 [56,57,61,62]. In these different studies this strain has been misrepresented as having a French origin, which is not the case. We confirm here that this strain was indeed isolated in the framework of this study  [60] regions are probably due to variety in the degree of feline presence according to area and also to climatic conditions, which may influence the sporulation and resistance of T. gondii oocysts. Sheep included in the study are grazed from spring to autumn; therefore, cats and wild felids may be an important source of T. gondii contamination. Romania has a temperate continental climate, being semi-arid in the southeast of the country and more humid in the regions inside the Carpathian Arc. This may explain the lower seroprevalence in the southeast of Romania. In Serbia, Klun et al. [39] found that region and farm type were associated with T. gondii seroprevalence. The MAT (modified agglutination test) is a serological technique used for the detection of anti-T. gondii antibodies in sheep [40,41]. We have isolated 18 T. gondii strains from lambs aged between 2.5 and 4 months with MAT titers ≥ 1:200. Dubey et al. [40] and Dumètre et al. [42] have obtained viable T. gondii strains from lambs aged between 6 and 12 months and ewes with MAT titers ≥ 1:50. Therefore, 53 T. gondii isolates of 68 lambs' myocardia (77.9%) and 8 T. gondii isolates out of 30 ewes' myocardia (26.6%) were obtained in the USA and France, respectively [40,42]. Likewise, in Spain, from ewe myocardial tissue, 20 (40%) T. gondii isolates were obtained out of 50 isolation attempts [43]. The difference between the rates of isolation of the parasites can be explained by the age of tested animals (lambs or ewes), the tissue used for bioassay (diaphragmatic tissue or myocardium), the quantity of tissue used for bioassay (5-25 g diaphragmatic tissue or 50 g myocardium) and the number of mice use for bioassay (2, 5 or 10) [40]. Moreover, a significant correlation was observed between the MAT titer and the percentage of T. gondii isolates obtained. If the MAT titer is > 1:16, the probability of obtaining isolates is about 65%. The probability decreases significantly when the MAT titer is < 1:16 [41].
In the present study, 18 strains could be isolated. Six of these 18 isolates could be genotyped using microsatellite markers, obtaining two complete genotypes (TgRO-1LCj; TgRO-2LCj), and four others incomplete with between 11 and 14 markers obtained (TgRO-3LCj; TgRO-4LCj; TgRO-5LCj; TgRO-6LCj). These six genotypes were identical on all markers obtained and belonged to the type 2 lineage. Considering that all six strains came from lambs reared in the same flock from Cluj County, it is probably the same epidemic clone. To our knowledge, this is the first study in Romania to report the isolation and characterization of viable T. gondii from seropositive lambs reared in an extensive growth system.
The genetic characterization of T. gondii strain circulating in Europe has shown that type II is by far most frequently found in livestock, followed by type III and type I [22,[43][44][45]. Neighbor-joining tree of T. gondii strains from Romania, inferred from calculated Cavalli-Sforza distances for 15 microsatellite markers. The circles indicate the Romanian strains (n = 8) with two black circles corresponding to the two strains of this study presenting a complete genotype; the squares correspond to the reference strains (type I in red, type II in green and type III in blue) The techniques used for diagnosis of T. gondii in sheep abortions can be serological, histological, molecular and biological. Toxoplasma gondii genomic DNA detection in various sheep abortion tissues is considered useful and demonstrates congenital transmission of the parasite [46,47]. Congenital infections occur only when the sheep becomes infected during the pregnancy. To associate T. gondii with reproductive disorders, it is necessary to identify the T. gondii-like lesions by histological examination and not only to detect the DNA of T. gondii [12]. Embryonic death, fetus absorption, fetal death, mummification, abortion and stillbirth are the consequences of the development of macroscopic lesions of the placenta, caused by focal inflammation and necrosis of the cotyledons [48,49]. However, the isolation of the parasite by bioassay in mice is the "gold standard" in the diagnosis of toxoplasmosis in sheep abortions [50,51]. The bioassay in mice also has disadvantages, such as ethical aspects and logistical and cost difficulties. Furthermore, its implementation in the routine diagnosis of congenital diseases in sheep is complicated.
In a recent meta-analysis that included global studies on T. gondii infection in sheep abortions, a prevalence of 42% by PCR and 16% by serology was reported [52]. The presence of T. gondii DNA in sheep fetuses was reported in various studies from Europe, with prevalence ranging between 5.4% in Spain (4/74) [53] and 53.7% in UK [54]. In the present study, 9 (11.8%) out of 76 tested aborted sheep fetuses were found positive by PCR, the DNA being detected in both brain and heart samples. In Spain, Fernández-Escobar et al. [43] found T. gondii DNA in 60.3% of fetal brain tissues, placenta tissue and brains from dead weak lambs, originating from 22 abortion outbreaks. However, the prevalence of parasite DNA was different depending on the type of tissue tested: 63.2% in fetal brains, 69.0% in placenta and 11.1% in brains of weak lambs, respectively [43]. Chessa et al. [55] also reported a variable prevalence of T. gondii DNA, with values of 3.5% in placenta, 87% in brain and 66.6% in liver samples from sheep fetuses.

Conclusions
Toxoplasma gondii remains a challenge for both human and veterinary medicine. All the knowledge related to the parasite biology, its genetic diversity, risk factors, immune response and epidemiology should be used to reduce the contamination of the environment as well as the transmission of the parasite. The present study reports a high prevalence of T. gondii infection in sheep and lambs in Romania and highlights therefore that human contamination by consumption of raw or undercooked sheep/lamb meat is an important risk factor. The study also emphasizes that the role of T. gondii as etiological agent associated with abortion and stillbirth in sheep should not be forgotten.

Availability of data and materials
The data supporting the results and conclusions of this article are included within the article.